Filamentous fungi produce a vast universe of secondary metabolites (SM), which display a broad range of useful activities for pharmaceutical and agricultural purposes, e.g. antibiotic, immunosuppressant, lipid lowering, or antimicrobial properties. One group of metabolites of particular interest are non-ribosomal peptide synthetase- (NRPS-) derived metabolites which have provided important leads for drug design. However, production of NRPS-based pathways is often very low (or undetectable) under laboratory conditions, in large part due to complex endogenous regulatory networks that control the biosyntheses of these metabolites, which remain, at best, poorly understood. As a result, many gene clusters containing NRPS or NRPS-like genes have no known metabolites (orphan gene clusters), even in well-studied species such as the genetic model Aspergillus nidulans. This R01 proposes in Aim 1 to identify metabolites associated with all remaining orphan NRPS and NRPS-like gene clusters in A. nidulans and the opportunistic pathogen A. fumigatus using an inducible promoter activation strategy coupled with comparative metabolomics based on high-resolution MS and 2D NMR spectroscopy. In Aim 2 we will utilize a Bacterial Artificial Chromosome (BAC)-based strategy to identify metabolites for all NRPS and NRPS-like gene clusters of A. terreus, an industrially important fungal species. A final goal of this aim is to examine the hypothesis that specific genes contained within a fungal NRPS/NRPS-like cluster may be predictive of the biological activity - and drug discovery potential - of the SM products. Overall, this parallel study of different species and comparison of different expression approaches will significantly advance understanding of NRPS-pathways and the scope of heterologous expression. Moreover, this work will provide advanced fungal lines for the community for SM cluster expression, a comprehensive assessment of A-domain substrate preference for fungal NRPS and an innovative methodology to identify new metabolites for biological evaluation.